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The Synthesis and Evaluation of Organometallic Dinuclear Ruthenium Complexes as Anti-Cancer Drugs

Fact Sheet

Reporting

Objective

"Current platinum-based anti-cancer drugs, such as cisplatin, are well established in the successful treatment of various forms of cancer, however, the use of these drugs is significantly limited by associated side effects and drug resistance. Ruthenium-based alternatives, some of which are in clinical testing, have shown considerable promise, exhibiting low general toxicity alongside selectivity and high activity toward cancerous tissue.

This proposal takes advantage of these remarkable properties in the design, synthesis and evaluation of novel dinuclear ruthenium-based anti-cancer drugs. These complexes will contain two ruthenium moieties linked by an organic tether – the structure of which will have a major influence on the conformation of the complexes and will therefore influence their interactions with biomolecular targets. The relationship between complex structures and their anti-cancer properties will be studied and their biomolecular targets identified, allowing the rational development of more potent anti-cancer drugs that express their activity by non-DNA damaging modes of action – essential in overcoming the toxicity and resistance problems of established metal complexes.

To achieve these objectives the candidate will build upon his experience in synthetic and analytical chemistry to isolate and characterize the complexes; he will develop expertise in bio-analytical techniques to allow him to identify their biomolecular targets and evaluate complex-biomolecule interactions/reactivity to probe their biological mode of anti-cancer activity.

Through this project the candidate will gain substantial new expertise in biochemistry, proteomics and advanced mass spectrometry. He will gain experience in the management and coordination of an independent project whilst developing scientific and complementary skills, such as leadership qualities and teaching skills - essential in his development as a promising independent researcher."

The continuing search for drugs that possess greater efficacy, are more selective, suffer from lower incidences of resistance and exhibit lower general toxicity is essential against the backdrop of figures showing that the number of people worldwide who die from cancer is expected to increase over the next 25 years and that one in three Europeans will develop cancer during their lifetime.

In the context of developing metal-based drugs for the treatment of cancer, platinum complexes have received great attention – stemming from the success of cisplatin (and derivatives). However, the use of cisplatin is limited by its associated side effects and drug resistance. To circumvent these problems, and in efforts to identify more selective compounds of increased activity, other metals have been utilized in the development of novel drug candidates. Ruthenium-based organometallic compounds are amongst the most promising alternatives and several families of such compounds have been evaluated and have shown diverse biological activity profiles. One of these, the RAPTA series of organometallic ruthenium(II) compounds, display selective activity on metastatic tumours in vivo and experimental evidence has shown proteins are major intracellular targets of these compounds, in contrast to the DNA damaging mechanism of action exerted by current platinum-based metallodrugs.

Taking advantage of previous research on the RAPTA series of compounds and current understanding of their mechanism of action, the main objectives of this project were:-• The development of dinuclear analogues of the RAPTA series of compounds, including conformationally rigid examples, to probe the influence of relative metal conformation on mode of action and biological efficacy.• Screening of the compounds in appropriate cell lines using MTT assays to enable an initial qualitative structure-activity analysis to be made.• Identification of molecular targets and characterization of complex-target interactions.

Work performed within the scope of this project took place in the Laboratory of Organometallic and Medicinal Chemistry (Prof. Paul J. Dyson) at EPFL. The results and progress achieved for each of the objectives is given below.

1. Synthesis of dinuclear organometallic Ru(II) compounds

In comparison to dinuclear platinum compounds there have been relatively few examples of dinuclear ruthenium compounds synthesised in the context of anticancer drug development, with no examples based on the RAPTA series being reported. However, from previously published work on mononuclear RAPTA compounds it was clear that the arene and PTA ligands may be further derivatized, thus providing a route toward the linking of two structures through covalent tethering of two arene or two PTA ligands.

Various synthetic routes were explored, ultimately allowing access to dinuclear organometallic ruthenium(II) compounds, tethered through the PTA or the arene ligands. For each family of dinuclear compounds, chiral linkers based on 1,2-diphenylethylenediamine (1,2-DPEN) were used that forced the RAPTA units into different relative conformations. In addition, analogous flexible linkers were also utilised. For the arene linked-compounds, crystallization conditions were found that provide crystals suitable for X-ray diffraction of dinuclear complexes with chiral 1,2-DPEN linkers (see inset), confirming the relative conformation of the two RAPTA units in these molecules were as predicted.

2. In vitro evaluation of cellular cytotoxicity.

All compounds were screened for cytotoxic activity toward several tumorigenic and non-tumorigenic cell lines using the MTT assay. For dinuclear compounds linked via their PTA ligands no relationship between linker structure and cytotoxicity was observed with all compounds exhibiting similar levels of moderate cytotoxicity toward all cell lines, indicative of no cellular selectivity. In contrast, for the arene-linked compounds, cytotoxic activity was observed to be dependent on the structure of the organic linker, with structures exhibiting a more closed conformation being significantly more active than those with a more open linear conformation. Furthermore, complexes with the closed conformation retained their high level of cytotoxicity in the cisplatin resistant A2780cisR cell line whilst complexes with the open conformation exhibited reduced cytotoxicity.

3. Identification of molecular targets and characterization of complex-target interactions.

The origins of the different cytotoxicities of the arene-linked dinuclear complexes was probed via coordination studies with various biological targets, including amino acids, nucleotides, peptides and oligonucleotides. In small molecule binding studies a clear relationship was found between complex conformation and the nature of adducts formed. Compounds with the closed conformation exhibited a higher proportion of 1:1 crosslinked adducts with 5'-GMP and L-histidine whilst for the more open structures a higher proportion of 2:1 ligand:complex adducts was observed. Binding studies with model peptides or oligonucleotides revealed all dinuclear complexes were able to crosslink these sequences through simultaneous binding of both ruthenium centres to amino acid/nucleotide residues. Further evaluation of peptide (H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-OH) crosslinking was achieved through electron-transfer dissociation (ETD) fragmentation of selected adducts (see inset). This study revealed coordination of the metal complexes to the histidine residues of the peptide in all cases. Given the dramatically different conformations observed for dinuclear compounds constructed with 1,2-DPEN ligands, it is reasonable to conclude that the tertiary structure of such peptide adducts must be different in each case.

Perspectives

Results from the research carried out within the scope of this Marie Curie fellowship have provided an intriguing insight into how the biological activity of dinuclear metal complexes may be influenced through control of complex conformation. The development of novel metallodrugs is a promising approach in the search for compounds with anticancer activity. This work feeds into this area by demonstrating that control of molecular conformation can modify complex-ligand interactions, ultimately leading to increased activity, and also drug resistance mechanisms operating in tumorigenic cells may be overcome.